The primate retina contains more than 17 classes of ganglion cells, but the contribution to vision of all but a few of these classes is unknown. This large gap in understanding is due to the fact that most ganglion cell types form such sparse mosaics that it is difficult with a single microelectrode or even an array of microelectrodes to record from enough cells of any given class to characterize its functional role. Another limitation of microelectrode technology is that the recording process is invasive, requiring penetration of the globe or, in the case of an eyecup preparation, enucleation of the eye. This precludes the ability to repeat experiments on the same cells and limits behavioral experiments on the same animals in which electrical responses have been obtained. However, rapid advances are being made in the development of reporter molecules that allow optical monitoring of the electrical responses of single neurons with multiphoton fluorescence. Moreover, the recent development of adaptive optics for correcting the eye's aberrations now makes it possible to image individual ganglion cells at ~ 2 micron resolution in the living primate eye. We will develop a new technology for retinal physiology, Functional Adaptive-optics Cellular Imaging in the Living Eye (FACILE) that combines adaptive optics in vivo imaging with optical recording to map the electrical activity of each of the several hundred ganglion cells simultaneously in a patch of monkey retina. We will use viral transduction to insert a genetically encoded calcium indicator (GCaMP3) into ganglion cells, exploring two delivery methods to further improve viral transduction of macaque ganglion cells: intravitreal injection of adeno- associated virus (AAV) in collaboration with John Flannery at UC, Berkeley and retrograde transport of pseudotyped equine anemia immunodeficiency virus (EAIV) injected into retino-recipient nuclei in collaboration with Ed Callaway at the Salk Institute. The development of FACILE will accelerate the complete characterization of the many pathways from the retina to the brain and will reveal the full contribution the retina makes to visual information processing. We will undertake early development of FACILE in a mouse model, and deploy the mature technology in monkey retina. In years 4-5, we will demonstrate the value of the approach by resolving the long-standing debate about whether the macaque retina contains direction-selective neurons, such as those that have been identified in the retinas of several other mammals. PUBLIC HEALTH RELEVANCE: The primate retina contains more than 17 classes of ganglion cells, but the contribution to vision of all but a few of these classes is unknown, a consequence of the weakness of existing physiological methodology for understanding novel cell types. This project will develop a new technology for retinal physiology, Functional Adaptive-optics Cellular Imaging in the Living Eye (FACILE) that combines adaptive optics in-vivo imaging with optical recording to map the electrical activity of each of the several hundred ganglion cells simultaneously in a patch of monkey retina. The novel approach will be used to examine the possibility that among the unknown ganglion cell classes are directionally selective ganglion cells, as in other mammalian retinas. This methodology will accelerate our analysis of the full contribution of the many pathways from retina to brain in primate visual information processing.